We established a nested PCR that amplifies the regulatory region of JC virus (JCV) DNA. Using this system, we detected JCV DNA from cerebrospinal fluid (CSF) samples from 19 patients strongly suspected of progressive multifocal leukoencephalopathy (PML). In one case, JCV DNA was detected until 5 months after the onset of PML, but was not detected between 5 and 11 months. The clinical symptoms of the patient appeared to be stabilized. In another case, JCV DNA was detected at high efficiencies in 2 and 3 months after the onset of PML, but was detected in 4 months only at a low frequency. The follow-up of this patient will be performed.The structures of JCV regulatory regions detected varied markedly. The 'typical ' regulatory regions in which domain A is deleted and domain B is duplicated accounted only for a fourth or third of the total regulatory regions detected. Some regulatory regions underwent more than two steps of rearrangements and some contained only deletions.We analyzed JCV regulatory regions in autopsy brains and other organs from PML patients. In one case, it was suggested that peripheral lymphocytes were preferentially infected with a JCV variant that carried one of the several rearranged regulatory regions in the brain. In another case, dual origins of JCV infection in the central nervous system were suggested.We reported that archetype JCV can efficiently replicate in COS-7, simian cells expressing SV40 T antigen. In this study, we examined the growth capacity of various PML-type JCVs in COS-7 cells. Some of the examined PML-type JCVs grew as efficiently as a representative archetype JCV (CY), whereas some replicated more slowly than CY. These data suggested that PML-type regulatory regions are variable in the capacity to enhance viral growth.